HLA HOMOZYGOUS INDUCED PLURIPOTENT STEM CELL (iPSC) LIBRARIES

ABSTRACT

The present specification provides libraries of HLA homozygous induced pluripotent cell lines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication 62/595,488, filed Dec. 6, 2017; the entire contents of whichare incorporated by reference herein.

BACKGROUND

The ability to culture and differentiate stem cells has offered greatpromise for the repair or replacement of damaged or defective tissuesand organs that has yet to be realized. The field of stem cell therapyoriginally grew out of research on embryonic stem cells. The developmentof induced pluripotent stem cells (iPSC) greatly expanded access to stemcells and reduced ethical concerns over their use. One remainingimpediment to clinical use of iPSCs, and cells differentiated from them,in the treatment of diseases and injuries is the potential forimmunological rejection of iPSC-derived tissue.

One potential solution is autologous donation. However, if there is agenetic component to the condition being treated, the autologous iPSCswould then carry the same defect(s). This would not be a concern inconditions for which a gene therapy was available—and indeed that is oneintended application of stem cell technology—but there are others whereno genetic therapy has been developed and still others that are complexmultigenic conditions not amenable to therapy by correction of theunderlying genetic defect(s). Another drawback to autologous donation isthe necessity of starting from scratch for every patient. The need toobtain tissue from a patient and induce pluripotency before initiatingthe steps of the actual treatment adds to the time before treatment cancommence and introduces opportunities for mishap, misadventure, andfailure of the process.

Allogeneic donation at need is also possible, but finding animmunologically compatible donor can be difficult and the potential needfor extensive immunosuppression is undesirable. And like autologousdonation, this approach also suffers from the need to start from thevery beginning of the process each time. Banking iPSC lines couldaddress this latter issue, but merely taking cells from willing donorswill exacerbate the problem of finding an immunologically compatibledonor, at least without assembling a massive bank of cells with diversetissue types. Some have proposed knocking out major histocompatibilitycomplex (MHC) genes to reduce or avoid immunological rejection, but thisresults in more highly manipulated cells which some will considerundesirable, especially if further genetic manipulation will be part ofthe therapeutic use. Also the cell will not be able to participate innormal immunophysiology.

Thus, there exists a need for off-the-shelf iPSC lines that areminimally manipulated, are capable of normal immunophysiologicinteractions, and can be used in a substantial portion of a patientpopulation. Herein disclosed is such an iPSC library.

SUMMARY

The herein disclosed embodiments include libraries of HLA (or MHC)homozygous induced pluripotent stem cell (iPSC) lines and methods forthe production of such cell lines and assembly of such libraries. Theindividual HLA homozygous iPSC lines of the libraries are useful forproviding cells for transplantation into HLA-matched recipients. TheiPSC line cells can be differentiated into a desired cell type or tissueprior to transplantation. A library itself provides a ready source ofHLA-matched donor cells for a substantial portion of a target, service,or recipient population, obviating the need to seek an individual donorat the time the need for a donation arises. By comprising HLA-homozygouscell lines the library can serve a much greater proportion of thetarget, service, or recipient population than if it the iPSC cell lineswere heterozygous.

Some embodiments constitute a library of from at least 15, 20, 25, 30 .. . or 100 to 1000, or any range bounded by integers therein, iPSC lineswherein the iPSC lines are homozygous for at least a selected set of MHCloci, and wherein each iPSC line in the library comprises a differentHLA (MHC) haplotype or a different combination of HLA (MHC) haplotypeand ABO blood type. It is preferred the iPSC lines be ABO type 0, but insome embodiments multiple iPSC lines may possess the same HLA (MHC)haplotype but different ABO types. In some embodiments the donor cell isnot ABO type 0, but the iPSC line is genetically engineered to be typeO. In some embodiments the selected set of comprises specifically HLAloci, while other embodiments comprise other MHC loci instead or inaddition. In some embodiments the selected set of comprise HLA-A, -B,and -DR. In some embodiments the selected set of MHC loci comprises atleast one locus from telomeric side of the major histocompatibilitycomplex or at least one locus from the centromeric side of the majorhistocompatibility complex, or both, in addition to or instead of thepreviously indicated loci. In some embodiments the selected set of locicomprises one or more of HLA-C, -DQ, or DP in addition to or instead ofthe previously indicated loci. In some embodiments the selected set ofloci comprises one or more of MICA or MICB in addition to or instead ofthe previously indicated loci. In some embodiments some or all of theiPSC lines are autozygous for the selected set of MHC loci.

In some embodiments the library comprises a certain proportion orpercentage of the most frequent haplotypes in a defined or referencepopulation or subpopulation. In various aspects of these embodiments thelibrary may comprise, for example, all haplotypes that occur at afrequency >2%, >3% or >4% in the defined population, or ≥50%, ≥60%,≥70%, ≥80%, ≥90%, or ≥95% of all haplotypes occurring at a frequencyof >1%, >0.9%, >0.8%, >0.7%, >0.6%, >0.5%, >0.4%, >0.3%, >0.2%, >0.1%, >0.09%, >0.08, >0.07%, >0.06%or >0.05% in the defined population, or combinations thereof. In variousembodiments the reference population is defined by geographic origin,geographic location, ethnic background, or a combination thereof.

In some embodiments at least 10 of the iPSC lines in the library have ahaplotype listed in Table 1

TABLE 1 HLA-A* HLA-B* HLA-DRB1* 01:01 08:01 03:01 03:01 07:02 15:0102:01 44:02 04:01 02:01 07:02 15:01 29:02 44:03 07:01 02:01 15:01 04:0101:01 57:01 07:01 03:01 35:01 01:01 02:01 40:01 13:02 30:01 13:02 07:0102:01 08:01 03:01 02:01 57:01 07:01 24:02 07:02 15:01 11:01 35:01 01:0133:01 14:02 01:02 23:01 44:03 07:01 01:01 07:02 15:01 02:01 15:01 13:0102:01 13:02 07:01 31:01 40:01 04:04 25:01 18:01 15:01 02:01 44:03 07:0102:01 44:02 13:01 02:01 44:02 01:01 01:01 08:01 15:01 03:01 07:02 01:0102:01 44:02 15:01 02:01 51:01 11:01 26:01 38:01 04:02

In some embodiments the homozygous haplotypes expressed by the iPSClines comprise at least those on the above list.

In some embodiments the iPS cell lines are derived from cord blood. Insome embodiments the iPS cell lines are derived from CD34⁺ cord bloodcells. In some embodiments the iPS cells are reprogrammed by, andinitially express, Oct-4, Sox-2, Klf-4, and c-Myc.

In some embodiments homozygous donors are identified by screening randomblood donors or random persons being tissue typed for other reasons. Inother embodiments homozygous donors are identified by screeningvolunteers from a general population. In still other embodimentshomozygous donors are identified by screening volunteers from apopulation likely to have a higher frequency of homozygosity or a higherfrequency of a particular desired haplotype.

Some embodiments constitute methods of producing a library of HLAhomozygous iPSC. Such methods can comprise screening whole blood,preferably cord blood for MHC homozygosity according to the parametersdescribed above, for genetic, cytogenetic and other genomic andchromosomal abnormalities, and for donor history and markers of exposureto infectious diseases. Such methods can further comprise isolatingwhite blood cells from the screened units of blood that were negativefor the genetic defects and exposure to the infectious diseases andpositive for the homozygosity, causing the white blood cells to expressOct-4, Sox-2, Klf-4, and c-Myc to form iPS cells, and culturing andcloning the iPSC to produce a population of cloned iPS cells homozygousfor the selected set of MHC loci. Such methods can further comprisecharacterizing the genotype of the iPSC by typing additional MHC locisuch as class I, II and III genes and including the A,B,C and DR loci ifthey were not part of the initial selected set. Such methods can furtherinclude screening the cells from iPSC clones for the presence ofchromosomal aberrations and evidences of genetic instability, andcryoprotecting and storing the cloned population of iPSC.

In aspects of the above methods the causing step can include usingvectors capable of inducing the target white blood cells to express theexogenous transcription factors Oct-4, Sox-2, Klf-4, and c-Myc. Suchvectors can be an episomal, viral, or a non-viral vector and thespecific transcription factors Oct-4, Sox-2, Klf-4, and c-Myc areencoded as DNA, RNA, or protein. Such methods can further compriseisolating hematopoietic CD34⁺ cells from the cord blood prior to thecausing step. Storing can comprises cryogenically preserving the iPSC attemperatures below −150° C.

Some embodiments constitute a library produced by the forgoing methods.

Some embodiments constitute methods for providing differentiated cellsderived from an iPSC library to a subject in need thereof. Such methodscan comprise determining the HLA haplotype for HLA-A, -B, -C and -DR ofthe subject; selecting an iPSC line from the library which contains amatch at all of HLA-A, -B, -C, and -DR loci with the subject's HLAphenotype for those loci; differentiating the iPSC into a cell typeneeded by the subject; and providing the differentiated cells to thesubject. Such methods can further comprise expanding and fullydifferentiating the iPS cells into a differentiated cell type (e.g.,neural cells, myocardial muscle cells, insulin-producing cells, etc.)and ensuring the disappearance of the pluripotent cells and inducingvectors, as well as the genetic stability of the differentiated cells.In some embodiments providing the differentiated cells to the subjectcomprises providing the differentiated cells to the subject's medicalprovider.

In one embodiment the library of HLA homozygous induced pluripotent stemcell (iPS) lines comprises at least 20 iPS cell lines homozygous foralleles at HLA-A, -B, -DR, MICA and MICB loci, each cell line expressinga different homozygous haplotype. In a further aspect of this embodimenteach of the iPS cell lines has been reprogrammed into pluripotentialityby means of non-endogenous transcription factors.

In one embodiment the library of HLA homozygous induced pluripotent stemcell (iPS) lines comprises at least 20 iPS cell lines homozygous foralleles at HLA-F and -DPB3 loci, each cell line expressing a differenthomozygous haplotype, wherein the haplotype difference can be at anHLA-A, -B, or -DR locus. In a further aspect of this embodiment each ofthe iPS cell lines has been reprogrammed into pluripotentiality by meansof non-endogenous transcription factors.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the cumulative percentage of the population possessingthe most frequent haplotypes for four US subpopulations (Caucasian,Hispanic, African-American, and Asian or Pacific Islander) based on theUS National Marrow Donor Program data.

FIG. 2 depicts the cumulative percentage of the population possessingthe most frequent haplotypes for three relatively homogenouspopulations: Japanese living in the same locale as their grandparents;Ashkenazi Jews in an Israeli Bone Marrow Donor Registry; and Germanblood donors. The data for Caucasians from FIG. 1 is also plotted forcomparison.

DESCRIPTION

Embodiments disclosed herein relate to libraries (also called arrays) ofinduced pluripotent stem cell (iPSC) lines that are homozygous for atleast major histocompatibility (MHC) antigens HLA-A, HLA-B, and HLA-DR,or at least HLA-A, HLA-B, HLA-C, and HLA-DR. These loci are the mostimportant to match between donor and recipient to gainimmunocompatibility and to facilitate engraftment of transplantedtissue. In alternative embodiments, the library of iPSC lines can becharacterized of defined as being homozygous at loci near either end ofthe major histocompatibility complex (MHC). For example, the constituentcell lines can be homozygous for at least one or more loci toward thetelomeric side of the MHC, such as HLA-F, MICE, HLA-90, MICG, HLA-G,MICF, HLA-K, HLA-U, or HLA-A, and for at least one of more loci towardthe centromeric side of the MHC, such as HLA-DQA1, HLA-DQB1, HLA-DQB3,HLA-DQA2, HLA-DQB2, HLA-DOB, HLA-Z, HLA-DMB, HLA-DMA, HLA-DOA, HLA-DPA1,HLA-DPB1, HLA-DPA2, HLA-DPB2 or HLA-DPB3. In further embodimentsaccording to either alternative, the iPSC lines are also homozygous atMICA (MHC class I chain-related protein A) and/or MICB (MHC class Ichain-related protein B).

Although the iPSC lines may be selected or defined according to the lociindicated above, in some embodiments the cell lines will becharacterized at additional loci: for example class I loci such asHLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, HLA-L, HLA-P, or HLA-V;and/or class II loci such as DRA, DRB2-DRB9, DQA1, DPA1, DPA2, DPB1,DPB2, DMA, DOA, or DOB; and/or MICA or MICB; and/or class III loci suchas C2, C4A, C4B, and CFB (the C-3-activating complement components),CYP21, and TNX. In some embodiments, the iPSC lines are homozygous atone or more of these additional loci. In some embodiments homozygosityat one of more of these loci is a selection criterion for inclusion in alibrary or part of the minimal definition of the iPSC lines in thelibrary.

In some embodiments the donors and resulting iPSC lines are not merelyhomozygous for the specified haplotype, but autozygous. That is, thehomozygosity reflects inheritance of both haplotypes from the samecommon ancestor as can result from consanguineous mating. Autozygositywill insure a greater degree of identity between the two haplotypes thanrandom homozygosity which allows for greater accumulation of mutationand greater likelihood of meiotic cross-over causing non-identity atunevaluated loci. In clinical genetics consanguineous unions are thosebetween persons related as second cousins or closer or having aninbreeding coefficient (the proportion of loci at which the offspring isexpected to receive identical alleles from both parents) 0.0156.

It is preferred that the donors of the homozygous cells be from ABOblood group O (so-called universal blood donor type), It is alsopreferred that the donors of the homozygous cells be male to avoidpotential reactivation of the one X-chromosome that is normallyinactivated in females. Thus in various embodiments at least 50, 60, 70,80, 90, 95, or 100 percent of the iPSC lines in a library are derivedfrom blood group O donors, from male donors, or from male, blood group Odonors.

By being homozygous, the cell lines express only one antigen for each ofthe loci and thus there is only one antigen per locus to match to thepotentially two expressed antigens per locus of prospective recipients.Or from the perspective of the prospective recipients, they need onlyfind an iPSC line in the library that expresses either of the antigensthey express at each locus. This greatly increases the likelihood offinding a match as compared to matching with heterozygous donors, inwhich case one is attempting to match two antigens at each locus. For ahematopoietic graft using neonatal cells, the National Cord BloodProgram estimates that to provide acceptable matches for 80-90% of theUS population, an inventory of 150,000 (random, primarily heterozygous)cord blood units would be needed. By contrast, based on data from theNational Marrow Donor Program, a library of homozygous iPSC linesrepresenting the 1000 haplotypes most commonly encountered in the USCaucasian population would provide a haplotype match for over 80% ofthat population. The likelihood of any random prospective recipientfinding a compatible cell line in the library is increased by theinclusion of HLA types or alleles, and HLA-haplotypes, which occur morefrequently in the prospective recipient population. The likelihood ofany random prospective recipient finding a compatible cell line in thelibrary is also increased by the inclusion of ever larger and morediverse set of iPSC lines. However, a point of diminishing returns isreached both with respect to the cost of assembling and maintaining thelibrary, and to the difficulty of finding (homozygous) haplotypesrepresenting ever rarer HLA types or haplotypes. No such library ofpractical size will ever contain a matching cell line for every possibleprospective recipient.

The assembly of a library of homozygous cells is facilitated by routineaccess to HLA-typed tissue, although a dedicated screening program couldbe used instead of, or in conjunction with, programs conducted forindependent reasons. Example 5 below describes assembling a library fromcord blood units from the National Cord Blood Program of the New YorkBlood Center. However any tissue typing lab, such as those associatedwith organ transplant centers, will in the course of their work, comeacross persons who are homozygous at their HLA loci. With properconsent, already donated blood, or blood or other tissue specificallydonated for this purpose, can be used as source material for thegeneration of iPSC lines.

The art of tissue typing has evolved since the first descriptions in1958 of what became known as human leukocyte antigens (HLA). Initiallytyping was done serologically using polyclonal antisera frommultitransfused patients and multiparous women. Over time, monoclonalantibodies were also adopted and now most typing is done at the geneticlevel, utilizing PCR, DNA hybridization, and sequencing. As dataaccumulated, it became clear that some serologic reactivities arose fromallelic variation at the same locus while other reactivities reflectedindependent loci. As more specific serologic reagents became available,more serologic types were identified and some types “split”; forexample, A9 became A23 and A24 and the A9 designation ceased to be used.Not surprisingly, the use of terminology has evolved along with ourunderstanding and technology and usage over time is not consistent. Weshall observe the following conventions:

-   -   HLA [antigen] identities defined by serologic reactivity shall        be referred to as HLA types, and in some contexts as serologic        types or serotypes, or phenotypes.    -   HLA [antigen] identities defined by DNA sequence shall be        referred to as alleles or genotypes.    -   A group of alleles or serotypes encoded on a single chromosome        shall be referred to as a haplotype.    -   A group of alleles or serotypes encoded at one or more loci, but        on both members of the chromosome pair, shall be referred to as        a tissue type.

It should be understood that some genotypic differences are silent withrespect to amino acid sequence and that others, while giving rise to adifference in amino acid sequence, do not generate a difference inserologic reactivity or, more to the point, in clinicalimmunoreactivity. Thus, such allelic variation is phenotypically silent.Conversely, some genotypic differences encode differences in amino acidsequence that can give rise to clinical immunoreactivity despite fallingwithin the same traditional HLA type. Depending on the particular use towhich the iPSC will be put, such allelic differences in immunogenicitymay or may not require consideration in determining a match.

The current convention for the naming of HLA alleles, set by the WHONomenclature Committee for Factors of the HLA System, are based on theresults of typing at the DNA sequence level are summarized as follows:

-   -   Exemplary HLA allele name: HLA-A*02:101:01:02N    -   Locus designation: The first field of the name refers to the HLA        locus (e.g., HLA-A) which is followed by an asterisk (*)        separating it from the allele designation.    -   Allele designations: each HLA allele name has a unique number,        composed of up to four sets of two or three digits separated by        colons. The length of the allele designation (number of        two-digit or three digit sets) is dependent on the sequence of        the allele and that of its nearest relative.    -   All alleles receive at least a four digit name, including the        first two sets of digits (fields) following the asterisk, longer        names are only assigned when necessary. The digits in the first        field (i.e., 02) describe the type, which often corresponds to        the serological antigen encoded by the allele at the locus (the        allotype). The second field lists the subtypes, numbered in the        order in which the respective DNA sequences have been determined        (i.e., 101). DNA sequences of alleles whose numbers differ in        the first two fields must differ in ways that change the amino        acid sequence of the encoded protein. Although not strictly        proper, it is a common practice to omit the colon between the        type and subtype fields when the subsequent fields are not used.        (02:101 and 02101 refer to the same allele).    -   Alleles that differ only by synonymous nucleotide substitutions        (also called silent or non-coding substitutions) within the        coding sequence are distinguished by the use of a third set of        digits (i.e., 01).    -   Alleles that only differ by sequence polymorphisms in the        introns, or in the 5′ or 3′ untranslated regions that flank the        exons and introns, are individualized by the use of a fourth set        of digits (i.e., 02).    -   In addition to the unique allele number, optional suffixes may        be added to a particular allele to indicate variations in its        expression status. Thus, alleles shown NOT to be        expressed—‘Null’ alleles—have the suffix ‘N’. Alleles that have        been shown to be alternatively expressed may have the suffix ‘L’        (low cell surface expression), ‘S’ (‘soluble’ encodes a secreted        protein not present at the cell surface), ‘C’ (is expressed at        the ‘cytoplasm’ but not at the cell surface), ‘A’ (‘aberrant’        when actual expression is in doubt) and ‘Q’ (when the expression        is ‘questionable’ because it resembles mutations seen in other        alleles that have abnormal expression. Because of the practical        importance of the change in expression, particularly with Null        alleles, the phenotype should be defined when transplantation is        contemplated.

The closely-linked alleles of the HLA genes form “haplotypes” thatremain together, segregating as a block in meiotic divisions, althoughrarely (frequency ˜0.8% between HLA-A and -B; and ˜1% between HLA-B and-DRB1) the HLA loci in a haplotype become separated by a cross-overevent leading to genetic recombination. Despite these crossovers, someof the alleles at different HLA loci “associate” with each other so thattheir frequency together in the same haplotype is higher, or muchhigher, than should be expected given their respective populationfrequencies. This phenomenon has been designated “LinkageDisequilibrium” (LD). Because the frequency of some specific HLAhaplotypes is much higher than expected from the frequency of therespective alleles, the frequencies of any haplotypes that include oneof the alleles involved in such a preferential association—but not theothers—are lower than expected under the assumption of randomassociation of alleles into HLA haplotypes. Many of the alleles at HLAloci, and the preferential haplotypes they integrate, have verydifferent frequencies in different ethnic groups within populations.This has contributed to the disproportionately higher access to matchedunrelated donors for patients of majority ethnic groups in the US.Because of the close distance between the HLA loci and low recombinationrate, and other causes not specifically understood, LD persists despitethe existence of recombination within the HLA haplotypes and includesspecific alleles of other gene loci located between and outside the“classic” Class I and Class II loci. In general, numerically, LD isdefined in relation to the quantity “D”:

DAB=pAB−pA pB

which is the difference between the frequency of a haplotype carryingboth alleles A and B at the two linked loci (pAB) and the product of thefrequencies of those alleles (pA and pB). Consequently, there is nodisequilibrium when DAB=0, and maximum disequilibrium (DAB=1) when atleast one of the four possible haplotypes (AB, Ab, aB, ab) does notoccur. It has been shown that, for many HLA haplotypes, much of theirpopulation frequency is due to LD, and that it extends to further lociin the adjacent areas of the chromosome. In terms of the array ofHLA-homozygous iPSC lines disclosed here, LD contributes two criticaladvantages, a) it allows for relatively easier assembly of iPSC lines,each with an HLA haplotype of comparatively high frequency, whichpermits a large fraction of the population to have access to matchedtissues derived from relatively few iPSC lines, and b) it provides ahigh level of certainty that alleles at additional MHC loci in thehaplotype (including non-classical HLA genes and MICA) are alsohomozygous, in LD with the HLA loci and very likely also matched to anyrecipients who are matched for the classical HLA loci.

Numerical aspects of linkage disequilibrium are presented below in Table2, refer to allele and haplotype frequencies of the three most frequentHLA haplotypes in US Caucasoids.

TABLE 2 Linkage Disequilibrium: the three most frequent Caucasoid HLAHaplotypes, the HLA alleles involved and their respective rankedfrequencies HAPLOTYPE: HLA-A*01:01; B*08:01; DRB1*03:01 - [RANKED:1^(st)] Allele Frequency (Rank) A*01:01 0.172 (2) B*08:01 0.125 (2)DRB1*03:01 0.129 (3) Haplotype Frequency = 0.074; Expected (pA × pB ×pDRB1) = 0.0028 (26-fold increase) HAPLOTYPE: HLA-A*03:01; B*07:02;DRB1*15:01 - [RANKED: 2^(nd)] Allele: Frequency (Rank) A*03:01 0.143 (3)B*07:02 0.140 (1) DRB1*15:01 0.144 (1) Haplotype Frequency = 0.035;Expected (pA × pB × pDRB1) = 0.0029 (12-fold increase) HAPLOTYPE:HLA-A*02:01; B*44:02; DRB1*04:01 - [RANKED 3^(rd)] Allele: Frequency(Rank) A*02:01 0.296 (1) B*44:02 0.090 (3) DRB1*04:01 0.091 (5)Haplotype Frequency = 0.026; Expected (pA × pB × pDRB1) = 0.0024(11-fold increase)

Thus, most of the population frequency of the most frequent haplotypesis due to LD and not to the high gene frequencies of the specificalleles. The other benefit of LD pursuant to using HLA homozygous graftscomes from the LD with, and homozygosity of, other genes in the MHCregion of chromosome 6, including MICA and MICB, as well asnon-classical HLA loci and many other genes. Non-HLA immune rejectiondue to humoral anti-MICA antibodies, for instance, is a distinctclinical problem in kidney transplantation. Products of somenon-classical HLA loci appear to influence immune regulation, others areimportant in regards defining the modulation of NK (natural killer) cellfunction and some were shown to increase the risk and severity of graftvs host complications in hematopoietic stem cell transplantation.

In determining a match, one generally will be matching the tissue type(both alleles at each of the loci under consideration) of theprospective recipient with the haplotypes (single alleles at each of theloci under consideration) present in the library. It is not necessary tomatch at the level of haplotype (though it would make matching atadditional MHC loci more likely and thus can be preferable) andhaplotyping prospective recipients is not routinely done as it requirestissue typing multiple blood relatives or specific cloning andsequencing procedures. Indeed, because of linkage disequilibrium mostmatches will in fact share a common haplotype. As long as theprospective recipient's tissue type includes a serotype or allele ateach locus that corresponds to the allele at the 3 (or 4 or 5 or more)specified loci in the haplotype then there is a match. In someembodiments matching is evaluated at the level of serotype (alsoreferred to as antigen-level resolution matching); for example ahaplotype containing an A*02:01 allele would be considered a match atthe HLA-A locus for any prospective recipient expressing any HLA-A2antigen. However, as more precise typing has become ever more commonthis has become an increasingly disfavored practice, though not entirelyabandoned. In other embodiments matching is evaluated at the level ofgenotype (also referred to as allele-level resolution matching); forexample a haplotype containing an A*02:01 allele would be considered amatch at the HLA-A locus for only a prospective recipient whose tissuetype included an A*02:01 allele; or in a variation of these embodiments,alleles differing only by genetically or immunogenically silentpolymorphisms are also considered a match. In some embodiments matchingis evaluated at the level of serotype for one locus and at the level ofgenotype at another locus; for example using antigen-level resolutionmatch for HLA-A and -B and allele-level resolution matching for DR.Clinical practice has largely moved to allele-level resolution matching,and it is generally associated with superior results in avoidingrejection. However, it should not be ignored that tissue and organtransplantation was successfully practiced before allele-levelresolution was even possible. Thus while the proportion of a populationthat would be served by a particular library might be calculated basedon allele-level resolution, actual clinical practice may proceed withless stringent matching—or even allow some mismatch—depending on theparticular application and other considerations that may arise in aparticular case. This would tend to increase the usefulness of anyparticular iPSC library.

The decision whether to use allele-level (genotype) or antigen-level(serotype) resolution in evaluating matching can be influenced by thesusceptibility of the organ or tissue to be repaired to rejection, andthus the necessity for more precise matching; for example kidney tissueis more susceptible to rejection than heart tissue which is moresusceptible than liver tissue. It can also be influenced by whether thelocation where the transplanted tissue is to be placed enjoys immuneprivilege; for example foreign antigens placed in the brain or eye aregenerally non-immunogenic. They are, however, still susceptible toimmune attack if a response to the antigen is induced elsewhere in thebody. The more susceptible the transplanted tissue is expected to be torejection, the greater the impetus to seek an allele-level match.However, while more stringent matches are generally expected to providebetter results, one may nonetheless decide to proceed with anantigen-level match if an iPSC providing an allele-level match is notavailable. The decision can also be influenced by the degree ofimmunosuppression, if any, that would be expected to be required tosupport engraftment and maintenance of the transplanted tissue and theacceptability of the immunosuppressive regiment for the particularprospective recipient. The least immunosuppression expected to beneeded—other things being equal—is preferred. In some instances thedecision to seek an allele-level match or accept an antigen level matchis made on a locus by locus basis.

If a perfect match at the desired level of resolution for the number ofloci being considered cannot be obtained, one can still consider goingforward with a mismatch. In some embodiments matching includesconsideration of the HLA-C allele or type, but as rejection is onlyinfrequently attributed to reactions against HLA-C, a mismatch at thatlocus can be deemed acceptable in some instances. The presence ofvarious defined antibody epitopes on the HLA molecule can also beconsidered so as to minimize the prevalence of antigenic and immunogenicsites on the mismatched HLA molecule as compared to those present in thetissue type of the prospective recipient (See EpiPedia of HLA posted onthe International HLA Epitope Registry website:wwwdotepregistrydotcomdotbr, which can be accessed by changing “dot” to“.”). The HLA Epitope Registry is also described in Duquesnoy, R J etal., Int J Immunogenetics 2012, 0, 1-6. Additional tools to understandand evaluate the presence of antibody epitopes are available on theinternet including: HLAMatchmaker (wwwdotepitopesdotnet), EpVix(wwwdotepvixdotcomdotbr), and the epitope frequency search function(wwwdotallelefrequenciesdotnet/hlaepitopes/hlaepitopesdotasp) at theAllele Frequency Net Database(wwwdotallelefrequenciesdotnet/defaultdotasp; Gonzalez-Galarza, F F etal., Nucleic Acid Research 2015, 28, D784-8).

In choosing an iPSC line from the library one can also consider thatrecipients can often tolerate maternal non-inherited HLA antigens. Thus,if the prospective recipient's mother's tissue type is known it can becombined with the prospective recipient's tissue type in evaluatingpotential matches to cell lines in the iPSC library.

In assembling a library of HLA homozygous iPSC lines, the baseconsideration is the availability of source material. One may work withwhat nature and happenstance provide or one may make a concerted effortto seek out donors and populations likely to have desired homozygoushaplotypes. Yet just because a homozygous cell is available, does notnecessarily make it appropriate for inclusion in a particular library.One should consider how common or rare the haplotype is, and how commonor rare the individual HLA types or alleles are. One must also considerhow the haplotype contributes to the overall coverage of the targetpopulation in combination with the haplotypes of the other likelymembers of the library.

HLA genes are not evenly distributed throughout the human population.Whether one is considering haplotypes or tissue types, their frequencywill vary from one target population to another. Thus it is important toconsider what target population the HLA homozygous iPSC line library isintended to serve. Populations can be defined in terms of geographic andnational borders, by ethnicity, or by a combination thereof. The librarycan be assembled to maximize coverage of a single target population, orit can be assembled to maximize coverage for multiple distinctpopulations found within a particular service area. Even if the libraryis constructed with one particular population in mind, it can be usefulto evaluate how well if covers other populations the library's servicearea. There will also be individuals who [are simply lucky and] will beable to find a compatible match despite not being part of the targetpopulation.

In evaluating HLA frequencies of the United States population, it hasbecome standard practice to stratify the population into for ethnicdivisions: Caucasian, African-American, Hispanic, and Asian or PacificIslander. HLA frequency data at the level of either genotype or serotypefor individual loci and for haplotypes is readily available on theinternet, for example, at the web site of the National Marrow DonorProgram (NMDP) (bioinformaticsdotbethematchclinicaldotorg). From aglobal perspective, HLA frequency data is typically aggregated bygeographic regions: North America; South and Central America, andCaribbean; Europe; North Africa; sub-Saharan Africa; Western Asia;Central Asia; North-East Asia; South Asia; South-East Asia; Australia;and Oceania (see the Allele Frequency Net Database, referenced above).However, the actual data is collected from a variety of studies of localnational and ethnic populations and medical databases, so it is possibleto compile frequencies of more narrowly defined populations as well (seefor example Eupedia's Distribution of HLA-A alleles by country;wwwdoteupediadotcom/genetics/HLA-A_allele_frequencies_by_countrydotshtml).

Haplotype frequencies for various US sub-populations are available fromthe NMDP (bioinformaticsdotbethematchclinicaldotorg). They havecatalogued 26,447 three-locus haplotypes, for HLA-A and HLA-B (class I)at antigen-level resolution and HLA-DRB1 (class II) at allele-levelresolution, across the four commonly used ethnic denominations:Caucasian, African-American, Hispanic, and Asian or Pacific Islander.The use of antigen-level resolution typing for the class I loci andallele-level resolution typing for the class II locus reflects, in part,the more extensive data and understanding of the serologic typing of theclass I loci as compared to the class II loci. Extensive typing of theclass II loci is more recent and has relied on DNA typing to a greaterextent. Thus the correlation between serologic type and gene sequence ismore robust for the class I loci and assignment to a serologic type fromsequence data alone can be made with greater confidence than for theclass II loci. It should be understood that different data sets based ondifferent sets of donors and/or different typing methods (e.g.,antigen-level vs. allele-level typing, etc.) will lead to differentobservations haplotype frequency within the same defined population.Such differences do not indicate that one or the other data set iswrong, but reflect only experimental variation and the fact that allsuch measures are approximations. As a general rule, the larger thesample size the more reliable and precise the frequencies will be.

The Caucasian subpopulation is the largest of these four subdivisions,constituting a majority of the US population, but is the least diversegenetically. More than 12,500 haplotypes with a frequency of at least1×10⁻⁶ per diploid genome have been observed. Eight haplotypes had afrequency >1%, three had a frequency over 2%, and the most commonhaplotype had a frequency >6%. Table 3 presents the 30 most commonhaplotypes observed in the Caucasian population in the NMDP data set,and their frequencies. For the Caucasian population, 25.5% and 29.7% ofindividuals would be predicted to have a matching haplotype in a libraryrepresenting the 20 or 30 most common haplotypes, respectively,evaluated at this level of resolution.

TABLE 3 Top 30 NMDP Caucasian Haplotypes Haplotype Rank A B DRB1Frequency 1 1 8 0301 0.062183 2 3 7 1501 0.030198 3 2 44 0401 0.020677 42 7 1501 0.019904 5 29 44 0701 0.015507 6 2 62 0401 0.012410 7 1 57 07010.011211 8 3 35 0101 0.011204 9 2 8 0301 0.008890 10 2 60 1302 0.00814311 24 7 1501 0.007100 12 2 57 0701 0.006801 13 2 44 0701 0.006729 14 3013 0701 0.006485 15 23 44 0701 0.006034 16 2 13 0701 0.005505 17 26 380402 0.005399 18 11 35 0101 0.005344 19 1 7 1501 0.005224 20 25 18 15010.004997 21 2 62 1301 0.004926 22 2 44 1501 0.004854 23 33 14 01020.004814 24 2 44 1301 0.004738 25 24 35 1104 0.004679 26 31 60 04040.004561 27 2 44 0101 0.004136 28 2 50 0701 0.004052 29 1 8 15010.003846 30 2 27 0101 0.003831

The Hispanic subpopulation is the second largest of these foursubdivisions. The NMDP observed more than 13,500 haplotypes with afrequency of at least 1×10⁻⁶ per diploid genome. Four haplotypes had afrequency >1%, but none over 2%. Table 4 presents the 30 most commonhaplotypes observed in the Hispanic population in the NMDP data set, andtheir frequencies. For the Hispanic population, 15.6% and 19.4% ofindividuals would be predicted to have a matching haplotype in a libraryrepresenting the 20 or 30 most common haplotypes, respectively,evaluated at this level of resolution.

TABLE 4 Top 30 NMDP Hispanic Haplotypes Haplotype Rank A B DRB1Frequency 1 29 44 0701 0.018354 2 1 8 0301 0.016801 3 2 35 0802 0.0156454 3 7 1501 0.011575 5 68 39 0407 0.008870 6 2 39 0407 0.007890 7 33 140102 0.007751 8 24 39 1406 0.007170 9 30 18 0301 0.006947 10 2 35 04070.006589 11 2 7 1501 0.005933 12 24 35 1104 0.005622 13 2 44 07010.005621 14 2 62 0802 0.004986 15 23 44 0701 0.004610 16 31 35 08020.004578 17 2 44 1301 0.004458 18 24 35 0407 0.004397 19 24 61 08020.004371 20 1 57 0701 0.004279 21 24 61 0407 0.004116 22 2 61 08020.003995 23 3 35 0101 0.003954 24 2 51 0802 0.003904 25 30 13 07010.003881 26 3 51 0701 0.003753 27 68 14 0102 0.003733 28 2 44 04010.003694 29 68 48 0404 0.003567 30 24 35 0802 0.003502

The African-American subpopulation is the genetically most diverse ofthese four subdivisions. The NMDP observed more than 13,500 haplotypeswith a frequency of at least 1×10⁻⁶ per diploid genome. Only twohaplotypes had a frequency >1%, and none were over 2%. Table 5 presentsthe 30 most common haplotypes observed in the African-Americanpopulation in the NMDP data set, and their frequencies. For theAfrican-American population, 10.3% and 13.2% of individuals would bepredicted to have a matching haplotype in a library representing the 20or 30 most common haplotypes, respectively, evaluated at this level ofresolution.

TABLE 5 Top 30 NMDP African-American Haplotypes Haplotype Rank A B DRB1Frequency 1 30 42 0302 0.014263 2 1 8 0301 0.011868 3 33 53 08040.007239 4 68 58 1201 0.007201 5 3 7 1501 0.006385 6 36 53 1101 0.0061517 34 44 1503 0.005485 8 2 44 0401 0.005146 9 30 42 0804 0.004910 10 6870 0301 0.004877 11 29 44 0701 0.004269 12 2 7 1501 0.003798 13 23 700701 0.003615 14 23 70 1101 0.003372 15 30 57 1301 0.003082 16 74 701302 0.003059 17 66 58 1503 0.002979 18 30 14 1503 0.002971 19 2 53 13020.002783 20 68 53 1503 0.002735 21 2 42 0302 0.002658 22 3 35 01010.002654 23 2 53 0804 0.002640 24 68 7 1503 0.002611 25 2 45 13020.002600 26 74 70 1101 0.002595 27 33 63 0102 0.002451 28 23 7 15030.002433 29 2 62 0401 0.002402 30 1 57 0701 0.002373

The Asian and Pacific Islander subpopulation is the smallest and has thegeographically most diverse ancestry of these four subdivisions. TheNMDP observed more than 11,500 haplotypes with a frequency of at least1×10⁻⁶ per diploid genome. Eight haplotypes had a frequency >1%, butnone were over 2%. The most common haplotypes observed in this group arelargely distinct from those of the other 3 subdivisions which haveseveral higher frequency haplotypes in common. Table 6 presents the 30most common haplotypes observed in the Asian and Pacific Islanderpopulation in the NMDP data set, and their frequencies. For the Asianand Pacific Islander population, 17.8% and 22.0% of individuals would bepredicted to have a matching haplotype in a library representing the 20or 30 most common haplotypes, respectively, evaluated at this level ofresolution.

TABLE 6 Top 30 NMDP Asian and Pacific Islander Haplotypes Haplotype RankA B DRB1 Frequency 1 33 58 0301 0.019323 2 33 44 0701 0.016077 3 2 460901 0.014848 4 24 52 1502 0.012177 5 33 44 1302 0.011553 6 30 13 07010.011410 7 33 58 1302 0.011260 8 1 57 0701 0.010689 9 11 75 12020.009466 10 24 7 0101 0.008143 11 24 35 1202 0.007326 12 2 46 08030.007228 13 1 37 1001 0.006629 14 11 62 0406 0.006346 15 24 54 04050.005637 16 24 38 1502 0.005567 17 29 7 1001 0.005333 18 24 75 12020.004551 19 2 60 0901 0.004451 20 26 8 0301 0.004399 21 11 13 15010.004397 22 11 46 0901 0.004236 23 2 61 1501 0.004028 24 2 13 12020.003921 25 24 60 0901 0.003744 26 24 61 0901 0.003527 27 2 61 09010.003506 28 11 38 1502 0.003317 29 11 62 1202 0.003293 30 2 75 12020.003245

While increasing the above haplotype sets from the 20 to the 30 mostcommon haplotypes in each population did increase the proportion of theprospective target populations that would have a matching haplotype, therate of increase declines after 10-20 of the top ranking haplotypes havebeen included (see FIG. 1). The rate of increase in the percentage ofthe indicated populations having at least one matching haplotype as morehaplotypes are added to the set is smallest for the African-Americanpopulation due to its greater genetic diversity. Conversely, theCaucasian population shows the greatest rate of increase due to itscomparatively restricted genetic diversity. Even with a library of 100iPSC lines harboring the 100 most common haplotypes, a Caucasian librarywould still provide haplotype matches for less than 45% of thecorresponding prospective recipient population and an African-Americanlibrary for just over 25% (see FIG. 1). Actual coverage to thepopulations would be somewhat better due to matching tissue typesarising from complementary, non-matching haplotypes, but thatcontribution is relatively modest (see Example 6). In addition to beinguncommon, such matches will generally be less satisfactory as well asthere are likely to be mismatches at non-evaluated loci in the MHC whilewith a true haplotype match the non-evaluated loci are also likely tomatch.

The United States population poses a particular challenge in assemblingiPSC line libraries that cover a substantial portion of the country'spopulation due to the degree of genetic diversity even within the foursubpopulations that the NMDP data is stratified into, and the limitedcorrelation of ethnic/genetic background with geographic location withinthe country. For countries or regions where there is a high correlationbetween ethnic/genetic background and geographic location (for example,Japan, Korea, the nations of Europe, those regions of China where thepopulation is predominantly Han Chinese, etc.) it is more feasible toassemble a library of limited size that will cover a substantial portionof a geographically- or nationality-defined population. It can beadvantageous to assemble multiple ethnically-focused libraries ofmoderate size—and without regard for geographic location—rather thanlarger, more universal libraries. To some extent this is an issue ofdistribution. For a single institution there may be little differencebetween assembling several smaller libraries or one large library. Evenif there is in effect a single, large library, ethnically-definedsub-libraries could be distributed to other institutions according tothe make-up of the population that institution serves. Indeed individualiPSC cell lines could be shipped elsewhere as needed. However, aninstitution assembling a library primarily for its own use, rather thanfor broader distribution, can afford to focus on the make-up of thelocal population that constitutes its principal patient pool.

Again using the Haplotype Frequency Search tool at the at the AlleleFrequency Net Database, the cumulative likelihood of matching at leastone haplotype in a library of the most common haplotypes for librarysizes up to 100 haplotypes was assessed for three relatively homogenouspopulations: Japanese living in the same locale as their grandparents(Japan pop 16; JAP), Ashkenazi Jews in Israel (Ezer Mizion Bone MarrowDonor Registry; AJW) and German blood donors (Germany pop 7; GER). Ingeneral, the cumulative percentage of the population rose more quicklywith increasing number of haplotypes, and tended toward leveling offmore slowly, than it did for even the NMDP Caucasian subpopulation (seeFIG. 2) further illustrating that the less diverse (more homogenous) thetarget population is, the greater coverage one can get with a givennumber of appropriately chosen haplotypes.

Cell lines that are homozygous for HLA-A, -B, -DR can be, and are evenlikely to be, homozygous at additional HLA loci. For example, the DQlocus is adjacent to the DR locus and there is a relatively small regionof DNA in which meiotic crossover would have to occur to separate thelinked alleles. Similarly, if homozygosity is observed for HLA-A and -Bthen homozygosity is likely for HLA-C as well. This is because the HLA-Clocus is between those of HLA-A and -B. While the distances from theHLA-B locus to the HLA-C locus and from the HLA-C locus to the HLA-Alocus are larger than that from the DQ locus to the DR locus, therewould need to be two meiotic crossover events, one between the HLA-Blocus and the HLA-C locus and one between the HLA-C locus and the HLA-Alocus, in order to maintain the linkage between HLA-A and -B whilesevering the linkage of those two loci to HLA-C.

In embodiments disclosed herein, the homozygous iPSC line librarycontains 20 cell lines, each harboring a unique haplotype. In otherembodiments the library contains 30, 40, 50 . . . 100 cell lines. Instill other embodiments the library contains up to 1000 cell lines.

In various embodiments, the library will be expected to provide a matchfor at least 5, 10, 15, 25, 30, 35, 40, 45, 50, 55, or 60 percent of atarget population. Likelihood of matching is evaluated withconsideration of HLA-A, -B, and -DRB1. The evaluation may furtherinclude any or all of HLA-C, -DP, -DQ, and additional -DR loci. Inaspects of these embodiments, the likelihood of providing a match isevaluated at antigen-level resolution for 0 to 6 loci. In furtheraspects of these embodiments the likelihood of providing a match isevaluated at allele-level resolution for 0 to 6 loci. In some aspects ofthese embodiments, the evaluation assesses likelihood of a match onlyconsidering likelihood of the prospective recipient having the samehaplotype as one of the library lines. In other aspects, the evaluationassesses likelihood of a match additionally considering the likelihoodof the prospective recipient having complementary, non-matchinghaplotypes for one of the library lines. In various embodiments,haplotype frequency for the target population is assessed in a data setof at least 2,000 samples, at least 4,000 samples, at least 10,000samples, at least 15,000, at least 20,000 sample, or at least 25,000samples.

A single library can be evaluated for coverage of a single targetpopulation or multiple target populations. In the former instance, thelibrary can target a single ethnic or geographic population. In thelatter instance, the library can target a geographic populationcomprising multiple distinct ethnic populations.

Target populations can be any ethnic group, any nationality, anygeographic region, or the service region of any institution providingstem cell therapies. Without limiting the breadth of the invention andpurely by way of example, ethnic groups can include Japanese, HanChinese, Koreans, Pacific Islanders, Native Americans,African-Americans, Hispanics, Caucasians, Europeans, Northern Europeans,Southern Europeans, Scandinavians, Poles, Slays, Germans, Italians,French, Russians, Ashkenazi Jews, Mizrahi Jews, Arabs, Persians, etc.Without limiting the breadth of the invention, and purely by way ofexample, regions can include North America; South and Central America,and Caribbean; Europe; North Africa; sub-Saharan Africa; Western Asia;Central Asia; North-East Asia; South Asia; South-East Asia; Australia;and Oceania; either as commonly used or as defined by the AlleleFrequency Net Database. Similarly, the term “region” can refer to ametropolitan area (for example, Greater New York City), a portion of acountry or other politically defined entity (for example, the MidwesternUS), or a geographic area (for example, the Mediterranean basin,Southeast Asia, or the Middle East).

In assembling a library, one can proceed only as homozygous cells becomeavailable. The likelihood of encountering a homozygous donor willreflect the frequency of the haplotype in the donor population. Thus,the use of homozygous donors biases in favor of high frequencyhaplotypes. Nonetheless it can be appropriate to consider whether anyparticular homozygous haplotype is a desirable addition to anyparticular library being assembled in light of the target population forwhich it is being assembled and the total number of cell lines thecompleted library is to contain. In many cases the donor and prospectiverecipient populations will be the same, but not necessarily. Thepopulation frequency of HLA haplotypes rapidly drop from the mostfrequent, thus becoming increasingly less likely to be encountered anddecreasingly important for enhancement of the iPSC array's effectivepopulation coverage. This is true with every ethnic group and makesfinding the next most frequent HLA homozygote progressively moredifficult and its expected contribution to the overall usefulness of theArray, smaller. Thus, adding HLA homozygous iPSC lines to the array mustbe a process mindful of each haplotype's frequency and of the effect ofits addition on the cost-benefit ratio. It is entirely acceptable, andpotentially preferable, for an iPSC library to not contain all of the10, 20, 30, 40, 50, or more, most common haplotypes in the population tobe served. As an array to serve a target population is assembled orexpanded, availability of an iPSC line will often be more important thanthe absolute rank of its haplotype in the target population. Thus itwill often make sense to include an iPSC line that is relatively commonin one population in arrays intended to serve other populations, inwhich the haplotype is substantially less common, due to itsavailability for inclusion and its relatively modest difference infrequency as compared to higher ranked haplotypes that are outside themost common haplotypes, for example the top 5 to 20 ranked haplotypes,for the targeted population. Especially once a basic iPSC line libraryis established, rather than expanding the array as more homozygous cellsare obtained, the homozygous cells can be stored frozen and onlyreprogrammed into iPSCs upon need for that haplotype.

The donor population and the service population will often be the same,but they need not be. In fact it can be advantageous to focus donorscreening efforts on more genetically homogenous subpopulations wherehomozygosity is more likely to be encountered. While the ranking ofallele frequencies may not reflect that of a broad or mixed servicepopulation, haplotypes that are very common in one subpopulation can beexpected to be present at a useful frequency among humans in general.

Example 1 discloses a panel of 20 homozygous haplotypes that can be usedto constitute an iPSC library targeting the US population generally. Oneof the haplotypes, A1, B8, DRB1*03:01, was the most common in theCaucasian (CAU) subpopulation and the 2nd most common in the Hispanic(HIS) and African-American (AFA) subpopulations, but rank only 36th inthe Asian and Pacific Islander (API) subpopulation (see Table 7). Thishomozygous haplotype would certainly be desirable to include in anylibrary targeted to the US population or the CAU, HIS, or AFAsubpopulations, but would be a more marginal choice for a libraryspecifically targeting the API subpopulation if the library was going tobe limited to only 20-25 iPSC lines. However, as explained above if, forexample, a haplotype ranked 15-25 were not available, this haplotypecould be included with minimal impact on the overall coverage of the APIpopulation despite ranking only 36th. If the API-targeted library wasgoing to contain 40 or more lines, then this haplotype would bedesirable to include. It would also be a good haplotype to include inany library targeting various Irish, English, Polish, German,Scandinavian, Italian, Spanish, Portuguese, and North African Jewishpopulations.

The same panel also contains the haplotype A33, B58, DRB1*03:01. Thiswas the most commonly observed haplotype in the API subpopulation andclearly would be appropriate to include in library of any size targetingthe API subpopulation individually or as part of a more generalpopulation. However, for the CAU, HIS, and AFA subpopulations this samehaplotype ranked only 668th, 1003rd, and 471st, respectively. Thus, itwould only become desirable to include this haplotype in a libraryspecifically targeting these subpopulations, individually or as a group,if the library were to contain several hundred cell lines.

In preferred embodiments the library will contain all haplotypesoccurring at a frequency of >2%, >3% or >4% in the targeted populationof prospective recipients. In preferred embodiments the library willcontain ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or all haplotypes occurring at afrequency of >1% in the targeted population of prospective recipients.In various aspects of these embodiments frequency shall be evaluatedbased on a data set for the targeted population containing at least 2000samples, at least 4000 samples, at least 10,000 samples, at least15,000, at least 20,000 sample, or at least 25,000 samples.

In some embodiments, a library of 30 iPSC lines will contain haplotypesproviding a match for at least 20%, 25%, 30%, 35%, or 40% of thetargeted population.

In some embodiments, an iPSC library with contain the five most commonhaplotypes for the targeted population. In some embodiments, an iPSClibrary with contain at least 10 of the 20 to 30 most common haplotypesfor the targeted population with each included integer and sub-rangeconstituting a distinct embodiment. In some embodiments an iPSC librarywill contain at least 10 to at least 20 of the 30 most common haplotypesfor the targeted population, with each included integer and sub-rangeconstituting a distinct embodiment. In some embodiments, an iPSC librarywill contain at least one third to at least two thirds of the mostcommon haplotypes within the total number of iPSC lines in the library,with individual fraction or sub-range constituting a distinctembodiment. Thus an iPSC line library containing 100 HLA homozygous celllines would include at least 34 to at least 67 of the 100 most commonhaplotypes in the targeted population, with each included integer andsub-range constituting a distinct embodiment. In further aspects ofthese embodiments, the library will include the 3, 4, 5, 6, 7, or 8 mostcommon haplotypes in the targeted population.

In further embodiments a library may be constructed as a hybrid of thesingle subpopulation focused libraries described above. For example, alibrary containing the 5 most frequent haplotypes in each of the CAU,HIS, and AFA—or CAU, HIS, AFA, and API—subpopulations could beassembled. Other defined subpopulations could be used instead in anynumber desired. Similarly, instead of the 5 most common haplotypes itcould be, for example, 4 of the 8 most common. Finally, the morepopulous or more genetically diverse subpopulation(s) of the group canbe over-represented. For example, a library could comprise 7 of the 10highest frequency haplotypes from the CAU subpopulation, 6 of the 10highest frequency haplotypes from the HIS subpopulation, 4 of the 10highest frequency haplotypes from the AFA subpopulation, and the 3 mostcommon haplotypes in the API subpopulation. The foregoing are onlyexamples and other libraries can be assembled according to theprinciples exemplified.

The following tables 7-10 provide the 40 most common haplotypes based onhigh resolution typing for HLA-A, -B, and -DRB1 in four USsub-populations: European Caucasian (EUR), African-America (AFA);Hispanic (HIS); and Asian &Pacific Islander (API). The alleledesignations in this table with the “g” suffix refer to allele groupsdefined in Table 2 (Maiers, M., Gragert, L., Klitz, W. High resolutionHLA alleles and haplotypes in the US population. 2007).

TABLE 7 EUR_rank A B DRB1 EUR_freq 1 0101 g 0801 g 0301 0.07404 2 0301 g0702 g 1501 0.03524 3 0201 g 4402 g 0401 0.02561 4 0201 g 0702 g 15010.02321 5 2902 4403 0701 0.01859 6 0201 g 1501 g 0401 0.01687 7 0101 g5701 0701 0.01371 8 0301 g 3501 g 0101 0.01275 9 0201 g 4001 g 13020.00970 10 3001 1302 0701 0.00954 11 0201 g 0801 g 0301 0.00896 12 0201g 5701 0701 0.00881 13 2402 g 0702 g 1501 0.00821 14 1101 g 3501 g 01010.00738 15 3301 1402 0102 0.00730 16 2301 g 4403 0701 0.00697 17 0101 g0702 g 1501 0.00675 18 0201 g 1501 g 1301 0.00674 19 0201g 1302 07010.00631 20 3101 4001 g 0404 0.00613 21 2501 1801 g 1501 0.00589 22 0201g 4403 0701 0.00585 23 0201 g 4402 g 1301 0.00565 24 0201 g 4402 g 01010.00530 25 0101 g 0801 g 1501 0.00515 26 0301 g 0702 g 0101 0.00508 270201 g 4402 g 1501 0.00503 28 0201 g 5101 g 1101 0.00488 29 2601 g 38010402 0.00473 30 0201 g 2705 g 0101 0.00462 31 0301 g 0801 g 0301 0.0044432 3002 1801 g 0301 0.00436 33 0201 g 1801 g 1104 0.00425 34 2402 g 0801g 0301 0.00425 35 2402 g 3502 1104 0.00416 36 0201 g 1501 g 0101 0.0036537 1101 g 0702 g 1501 0.00361 38 6802 1402 1303 0.00343 39 0201 g 5101 g1301 0.00343 40 0201 g 1501 g 1501 0.00341

TABLE 8 AFA_rank A B DRB1 AFA_freq 1 3001 4201 0302 0.01542 2 0101 g0801 g 0301 0.01169 3 6801 g 5802 1201 g 0.00782 4 6802 1510 03010.00713 5 3303 5301 0804 0.00697 6 3601 5301 1101 0.00676 7 0301 g 0702g 1501 0.00673 8 3402 4403 1503 0.00623 9 2902 4403 0701 0.00586 10 0201g 4402 g 0401 0.00539 11 2301 g 1503 g 0701 0.00501 12 7401 g 1503 g1302 0.00490 13 6802 0702 g 1503 0.00391 14 3001 4201 0804 0.00384 153002 1402 1503 0.00375 16 6802 5301 1503 0.00364 17 7401 g 5703 13030.00356 18 2902 4901 1503 0.00351 19 2301 g 4403 1503 0.00337 20 0201 g0801 g 0301 0.00300 21 0201 g 1501 g 0401 0.00297 22 6602 5801 g 15030.00293 23 0201 g 4501 g 1302 0.00289 24 6601 5802 1301 0.00287 25 2301g 1503 g 1503 0.00287 26 0201 g 0702 g 1101 0.00282 27 2301 g 5301 11010.00274 28 0201 g 4501 g 1503 0.00272 29 6802 5301 1303 0.00267 30 7401g 1503 g 1503 0.00264 31 2301 g 5301 0701 0.00250 32 2301 g 0702 g 09010.00246 33 0201 g 5101 g 1303 0.00245 34 2301 g 4201 0302 0.00240 356802 5301 1302 0.00235 36 0301 g 5802 0701 0.00234 37 2301 g 4403 07010.00232 38 2501 1801 g 1501 0.00230 39 2601 g 0801 g 1304 0.00227 406802 4201 0302 0.00227

TABLE 9 HIS_rank A B DRB1 HIS_freq 1 2902 4403 0701 0.01702 2 0101 g0801 g 0301 0.01538 3 0301 g 0702 g 1501 0.01293 4 3002 1801 g 03010.00823 5 3301 1402 0102 0.00788 6 6803 3905 0407 0.00652 7 2301 g 44030701 0.00636 8 2402 g 3906 1406 0.00595 9 0201 g 0702 g 1501 0.00587 100206 3905 0407 0.00552 11 0201 g 3517 0802 0.00545 12 2402 g 3502 11040.00504 13 0201 g 3512 0802 0.00448 14 0201 g 1515 0802 0.00445 15 0201g 4402 g 1301 0.00439 16 3001 1302 0701 0.00428 17 6802 1402 01020.00427 18 0201 g 0801 g 0301 0.00417 19 0201 g 4403 0701 0.00411 201101 g 2705 g 0101 0.00400 21 6801 g 4801 g 0404 0.00390 22 0101 g 57010701 0.00381 23 0301 g 3501 g 0101 0.00369 24 0301 g 5101 g 0701 0.0036525 0206 4002 g 0802 0.00362 26 0201 g 3501 g 0407 0.00359 27 0201 g 5101g 1101 0.00358 28 0101 g 0702 g 1501 0.00356 29 2402 g 4002 g 08020.00350 30 0201 g 1402 0102 0.00347 31 0201 g 3512 0407 0.00344 32 31013501 g 0802 0.00344 33 2402 g 4002 g 0404 0.00334 34 2501 1801 g 15010.00328 35 6801 g 4002 g 0407 0.00325 36 2402 g 3905 0407 0.00319 371101 g 5201 g 1502 0.00310 38 2601 g 3801 0402 0.00302 39 0201 g 1501 g0401 0.00302 40 0201 g 1801 g 0301 0.00300

TABLE 10 API_rank A B DRB1 API_freq 1 3303 5801 g 0301 0.02335 2 0207 g4601 0901 0.01597 3 3303 4403 0701 0.01499 4 3001 1302 0701 0.01466 53303 5801 g 1302 0.01434 6 1101 g 1502 1202 0.01216 7 2402 g 5201 g 15020.01022 8 0101 g 5701 0701 0.01002 9 3303 4403 1302 0.00866 10 0101 g3701 1001 0.00792 11 2901 g 0705 g 1001 0.00683 12 2402 g 4001 g 09010.00607 13 1101 g 4601 0901 0.00557 14 2402 g 5401 0405 0.00552 15 2402g 0702 g 0101 0.00550 16 1101 g 4001 g 0803 0.00514 17 2601 g 0801 g0301 0.00512 18 1101 g 3802 1502 0.00508 19 0207 g 4601 0803 0.00504 201101 g 1301 1501 0.00489 21 1101 g 5401 0405 0.00488 22 0201 g 1301 12020.00481 23 1101 g 1501 g 0406 0.00432 24 2402 g 4001 g 1501 0.00417 252407 3505 1202 0.00415 26 2402 g 4601 0901 0.00411 27 2402 g 5101 g 09010.00410 28 1101 g 4001 g 0901 0.00404 29 0201 g 4001 g 1101 0.00402 300203 3802 1602 0.00360 31 2402 g 4001 g 0403 0.00359 32 1101 g 3501 g1501 0.00348 33 2417 1502 1202 0.00341 34 1101 g 4001 g 1501 0.00339 352402 g 1301 1501 0.00335 36 1101 g 3901 g 0803 0.00334 37 2402 g 59010405 0.00332 38 1101 g 5201 g 1502 0.00325 39 0201 g 5101 g 0901 0.0032340 0101 g 0801 g 0301 0.00313

Further disclosed herein are induced pluripotent stem cells (iPSCs)formed from the identified HLA homozygous cells. The iPSCs can beproduced by any cell or tissue including, but not limited to, umbilicalcord blood, peripheral blood, bone marrow, adipose tissue, gonadaltissue, and others.

Stem cells are characterized by two distinct capabilities: self-renewalthrough mitotic cell division and the potential for differentiation intofunctional cells, tissues and organ. Thus, a critical quality of“sternness” is the unique ability to engage in “asymmetrical division”i.e., to divide producing two different cells: one, a stem cellidentical to its parent and the second, a cell with the capacity toengage in differentiation. There is a hierarchy of “sternness”, definedby the degree of limitation to the stem cell's capacity to generate anytype of cells. Thus, those emerging from the oocyte's first fewdivisions are totipotential stem cells, able to differentiate into anycell and tissue in the body—plus the extraembryonic, or placental,cells. Pluripotential stem cells, capable of forming the differenttissues of the embryo's three germ layers (endo-, meso- andecto-dermal), but not extraembryonic cells, and multipotential orprogenitor stem cells which give rise to only some cell linages, such asthe hematopoietic stem cells (HSCs) that originate the various celltypes of the blood and immune systems. Pluripotentiality is associatedwith the expression of functional “molecular markers”, such as, but notlimited to 5T4, ABCG2, Activin RIB/ALK-4, Alkaline Phosphatase/ALPL,E-Cadherin, Cbx2, CD9, CD30/TNFRSF8, CD117/c-kit, CDX2; CHD1,Cripto,DNMT3B, DPPA2, EpCAM/TROP1, ERR beta/NR3B2, ESGP, F-box protein15/FBXO15, FGF-4; FGF-5; FoxD3, GBX2, GCNF/NR6A1, GDF-3, Integrin alpha6/CD49f, Integrin beta 1/CD29, KLF4, KLF5, L1TD1, Lefty, LIN-28A,LIN-28B, LIN-41, c-Maf, c-Myc, Nanog, Oct3/4, Oct-4A, Oct-4B,Podocalyxin, Rex-1/ZFP42, Smad2, Smad2/3, SOX2, SSEA-1, STAT3, TBX2,TEX19, TRA-1-60(R), TROP-2, UTF1.

Pluripotent stem cell lines can be derived from embryonic murine (mESC)and human cells (hESC) cultured in-vitro, and have been extensivelystudied, despite the inherent ethical issues regarding the possibleclinical use of the latter, to generate differentiated tissues andcells. Differentiated human adult cells can be induced to become iPSCthrough the introduction of four transcription factors, Oct4 (Octamerbinding transcription factor-4), Klf4 (Kruppel-like factor-4), Sox2 (Sexdetermining region Y)-box 2, and c-Myc (avian myelocytomatosis viraloncogene [v-Myc] homolog). Although this combination of four factors hasbeen the most frequently reported for the production of iPSC, each ofthe factors can be functionally replaced by related transcriptionfactors, miRNAs, small molecules and non-related genes such as lineagespecifiers, and some even omitted—especially through changes in the p53gene—in some cases.

In one aspect, a method for preparing an iPSC is provided. The methodincludes transfecting a cord blood stem cell with nucleic acids encodingan Oct-4 protein, a Sox2 protein, a Klf protein, and a cMyc protein toform a transfected cord blood stem cell. The transfected cord blood stemcell is allowed to divide thereby forming the iPSC line. Other methodsof producing iPSC lines are within the scope of the present disclosure.

A “cord blood hematopoietic stem cell” refers to an adult stem cell thatresides in cord blood and is characterized by a lesser potency to selfrenew and differentiate than a pluripotent stem cell. Most hematopoieticstem cells express the CD34+ marker, including cord blood hematopoieticstem cells.

A “cell culture” is a population of cells residing outside of anorganism. These cells are optionally primary cells isolated from a cellbank, animal, or blood bank, or secondary cells that are derived fromone of these sources and have been immortalized for long-lived in vitrocultures.

The term “transfection” or “transfecting” is defined as a process ofintroducing nucleic acid molecules to a cell by non-viral or viral-basedmethods. Non-viral methods of transfection include any appropriatetransfection method that does not use viral DNA or viral particles as adelivery system to introduce the nucleic acid molecule into the cell.Exemplary non-viral transfection methods include calcium phosphatetransfection, liposomal transfection, nucleofection, sonoporation,transfection through heat shock, magnetifection, and electroporation. Insome embodiments, the nucleic acid molecules are introduced into a cellusing electroporation following standard procedures well known in theart. In some embodiments, the non-viral vector is an episomal vector ora modified RNA. For viral-based methods of transfection any useful viralvector may be used in the methods described herein. Examples for viralvectors include, but are not limited to retroviral, adenoviral,lentiviral, paramyxoviral (including Sendai viral), and adeno-associatedviral vectors. In some embodiments, the nucleic acid molecules areintroduced into a cell using a paramyxoviral vector following standardprocedures well known in the art.

There are multiple methods to generate iPSCs, including virus-mediatedgene transduction and chemical induction. While retroviral vectorsrequire integration into host chromosomes to express reprogramminggenes, DNA-based vectors such as adenovirus, adeno-associated virus, andplasmid vectors exist episomally and do not require integration;however, they may still be integrated into host chromosomes at certainfrequencies. Unlike these vectors, Sendai virus reprogramming vectors(paramyxoviral vectors) do not integrate into the host genome or alterthe genetic information of the host cell. Sendai virus (SeV) is arespiratory virus of mouse and rat, classified as mouse parainfluenzavirus type I belonging to the Paramyxoviridae family. SeV is anenveloped virus of 150-250 nm in diameter whose genome is a single chainRNA (15,384 bases) in the minus sense. Six genes coding for viralproteins are situated sequentially on the genome of the wild-type SeV inthe following order (starting from the 3′ end of the genomic RNA): (1)nucleocapsid protein (NP) which forms the core nucleocapsid complex withthe genome RNA; (2) phosphoprotein (P) which is the small subunit of theRNA polymerase; (3) matrix protein (M) which supports the envelopestructure from the inside; (4) fusion protein (F) which fuses the viralenvelope with cell membrane when the virus enters the cell; (5)hemagglutinin-neuraminidase (HN) which recognizes the cell surfacereceptor sialic acid; and (6) large protein (L) which is the largesubunit of RNA polymerase.

Because SeV infects cells by attaching itself to the sialic acidreceptor present on the surface of many different cells, it can infect awide range of cell types of various animal species. Activation of Fprotein by a protease is required for the virus-cell fusion process totake place. After infection, the virus goes through genome replicationand protein synthesis, and then daughter virus particles are assembledand released. Vectors comprising modified, non-transmissible forms ofSeV can safely and effectively deliver and express key genetic factorsnecessary for reprogramming somatic cells into iPSCs. Deletion of thegene encoding the F protein is renders the vector incapable of producinginfectious particles from infected cells. Desirable Sendai vectors arenon-integrating and remain in the cytoplasm. In addition, the host cellcan be cleared of the vectors and reprogramming factor genes byexploiting the cytoplasmic nature of SeV.

In certain embodiments, the cells are transfected with SeV vector(s)causing the target cells to express all of the transcription factorsOct-4, Sox 2, Klf4, and cMyc. Nucleotide sequences of these factors,within SeV expression vectors, are incorporated into the target cells,causing the cells to express all of the factors. In certain embodiments,the cells are transfected with a fragment comprising less than the fulllength gene. In such case, the fragment must induce the same activity inthe target cell as the full-length gene.

Oct-4 (octamer-binding transcription factor 4) also known as POU5F1 (POUdomain, class 5, transcription factor 1) is a protein that in humans isencoded by the POU5F1 gene. Oct-4 is a homeodomain transcription factorof the POU family. This protein is critically involved in theself-renewal of undifferentiated embryonic stem cells. As such, it isfrequently used as a marker for undifferentiated cells. Oct-4 has 4isoforms. The protein sequence (e.g., isoform 1, NCBI NP_002692.2) andnucleotide sequence (e.g., isoform 1, NCBI NM_002701.5) of Oct-4 arepublically available.

Sox2 (sex determining region Y)-box 2, is a transcription factor that isessential for maintaining self-renewal, or pluripotency, ofundifferentiated embryonic stem cells. A “Sox2 protein” as referred toherein includes any of the naturally-occurring forms of the Sox2transcription factor, or variants thereof that maintain Sox2transcription factor activity (e.g. within at least 50%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100% activity compared to Sox2). In someembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring Sox2 polypeptide. The protein sequence(e.g., NCBI NP_003097.1) and nucleotide sequence (e.g., NCBINM_003106.3) of Sox2 are publically available.

A “Klf4 protein” as referred to herein includes any of thenaturally-occurring forms of the Klf4 transcription factor, or variantsthereof that maintain Klf4 transcription factor activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto Klf4). In some embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring Klf4polypeptide. The protein sequence (e.g., isoform 1, NCBI NP_001300981.1)and nucleotide sequence (e.g., isoform 1, NCBI NM_001314052.1) of Klf4are publically available.

A “cMyc protein” as referred to herein includes any of thenaturally-occurring forms of the cMyc transcription factor, or variantsthereof that maintain cMyc transcription factor activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto cMyc). In some embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring cMycpolypeptide. The protein sequence (e.g., NP_002458.2) and nucleotidesequence (e.g., isoform 1, NCBI NM_002467.4) of Klf4 are publicallyavailable.

In certain embodiments, the Sendai virus vector is a non-replicativevector. An exemplary Sendai virus vector, while incapable ofreplication, remains capable of productive expression of nucleic acidsencoding protein(s) carried by the vector, thereby preventing anypotential uncontrolled spread to other cells or within the body of asubject. This type of Sendai vector is commercially available as aCytoTune™-iPSC Sendai viral vector kit (ThermoFisher Scientific).

Any method suitable to produce stable iPSC with no viral footprint andappropriate preservation of genetic characteristics are appropriate forthe reprogramming of HLA homozygous cells for inclusion in the disclosedarray or library.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids, or one or more polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of residues (nucleotides or amino acids) that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms or by manual alignment andvisual inspection (see, e.g., the NCBI web site or the like). Suchsequences are then said to be “substantially identical.” The definitionalso includes sequences that have deletions and/or additions, as well asthose that have substitutions. The identity exists over a region such asa region that is at least about 25 amino acids or nucleotides in length,over a region that is 50-100 amino acids or nucleotides in length, or aregion comprising the entire amino acid or nucleotide sequence asindicated.

Allowing the transfected cord blood stem cell to divide and therebyforming the iPSC may include expansion of the cord blood stem cell aftertransfection, optional selection for transfected cells andidentification of pluripotent stem cells. Expansion, as used herein,includes the production of progeny cells by a transfected cord bloodstem cell in containers and under conditions well know in the art.Expansion may occur in the presence of suitable media and cellulargrowth factors. Cellular growth factors are agents which cause cells tomigrate, differentiate, transform or mature and divide. They arepolypeptides which can usually be isolated from various normal andmalignant mammalian cell types. Some growth factors can also be producedby genetically engineered microorganisms, such as bacteria (E. coli) andyeasts. Cellular growth factors may be supplemented to the media and/ormay be provided through co-culture with irradiated embryonic fibroblaststhat secrete such cellular growth factors. Examples of cellular growthfactors include, but are not limited to FGF, bFGF2, and EGF.

Where appropriate, the expanding transfected cord blood stem cell may besubjected to a process of selection. A process of selection may includea selection marker introduced into a cord blood stem cell upontransfection. A selection marker may be a gene encoding for apolypeptide with enzymatic activity. The enzymatic activity includes,but is not limited to, the activity of an acetyltransferase and aphosphotransferase. In some embodiments, the enzymatic activity of theselection marker is the activity of a phosphotransferase. The enzymaticactivity of a selection marker may confer to a transfected cord bloodstem cell the ability to expand in the presence of a toxin. Such a toxintypically inhibits cell expansion and/or causes cell death. Examples ofsuch toxins include, but are not limited to, hygromycin, neomycin,puromycin and gentamycin. In some embodiments, the toxin is hygromycin.Through the enzymatic activity of a selection maker a toxin may beconverted to a non-toxin, which no longer inhibits expansion and causescell death of a transfected cord blood stem cell. Upon exposure to atoxin, a cell lacking a selection marker may be eliminated and therebyprecluded from expansion.

Identification of the iPSC may include, but is not limited to theevaluation of the afore mentioned pluripotent stem cell characteristics.Such pluripotent stem cell characteristics include without furtherlimitation, the expression or non-expression of certain combinations ofmolecular markers. Further, cell morphologies associated withpluripotent stem cells are also included in pluripotent stem cellcharacteristics.

Additional characterizations of the iPSCs can include, but are notlimited to, determination of colony and cell morphology in cultureincluding specific immunofluorescence and immunocytochemistry; sterilitytesting; Mycoplasma testing; presence of endotoxin, determination ofresidual reprogramming plasmid or factor expression; fow cytometric andimmunocytochemistry markers for embryonal germ layer, differentiation,and self-renewal; ability to form embryoid bodies (in vitro) andteratomas; histopathology revealing three-lineage differentiation;expression analysis (cRNA microarrays, Pluritest and hPSC ScoreCard);karyotyping (G-banding); copy number variation (CNV) and comparativegenomic hybridization (CGH); characterization of short tandem repeats(STR); and whole genome sequencing. One or more of thesecharacterization assays are performed on the HLA homozygous iPSC lines.

In certain embodiments, the HLA homozygous iPSC are maintained in acryopreserved library until such time as a need arises for a specificsample for a recipient. At that time the iPSC are thawed, cultured, anddifferentiated into the cell type needed by the recipient. Oncedifferentiated, the specific cells are then administered to therecipient.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification,

Example 1 Generation of iPSC from CD34+ Cord Blood Cells

Umbilical cord blood (UCB) is obtained from full-term deliveries afterreceiving informed consent. UCB is collected in bags containing heparinand processed within 24 hr by cryopreserving. The cord blood is HLAtyped and, if homozygous, the cord blood unit is suitable for inclusionin the library and for generation of iPSCs.

After separation over Ficoll Isopaque, low density cells are washed inmedia supplemented with 2% FCS. CD34+ cells are selected frommononuclear cell suspensions by immunomagnetic cell separation usinganti-CD34 antibodies. CD34+ cells are cultured for 5 days in mediumsupplemented with 10% FBS, human stem cell factor (SCF), humanthrombopoietin (TPO), human interleukin-3, rhIL-6, rhIL-6 receptor α,rhFlt3 ligand, human granulocyte macrophage colony-stimulating factor,and rhM-CSF.

Reprogramming of CD34+ UCB cells into iPSCs is performed 5 days afterinitial culture using SeV vectors encoding Oct-4, Sox2, Klf4, and c-Myc.The floating cells are discarded and the adhesive cells are treated with0.25% trypsin/EDTA and resuspended in the same medium described above.To transduce cells, SeV vectors containing the four separatereprogramming factors are added to the cell suspension. The fourreprogramming factors are included on 1, 2, 3, or 4 different SeVvectors. Vector-transduced cells are immediately plated onto a 12-wellplate precoated with 5 μg/cm² fibronectin (e.g., RetroNectin®,Clontech). The culture plate is centrifuged at 1000×g at 32° C. for 45min. The next day, medium is replaced with fresh medium. Two days later,cells are trypsinized and passed onto two 10-cm gelatin-coated culturedishes. The cultures are maintained in human embryonic stem cell (hESC)medium containing DMEM/F12, 20% KnockOut® Serum Replacement, 1% minimumessential medium (MEM) nonessential amino acids, 1 mM L-glutamine, 0.1mM β-mercaptoethanol, and 8 ng/mL of basic fibroblast growth factor(bFGF). iPSC colonies are manually isolated based on morphology betweenday 14 to day 30 postinfection. iPSC cultures are further maintained onplates coated with MATRIGEL® (BD Biosciences).

Example 2 Teratoma Formation by Human iPSCs

iPSC clones generated in Example 1 are tested in a teratoma assay. iPSCsat a concentration of 1×10⁶/125 μl are injected subcutaneously into theflank of a severe combined immunodeficiency mouse. After 75 days, themice are euthanized, evaluated for gross evidence of tumor formation,and representative sets of tissues, such as liver, kidneys, spleen,pancreas, adrenal gland, gall bladder, lungs, heart, brain,gastrointestinal tract, urogenital tract, and left hind limb arecollected for histology. Collected tissues are fixed in 10%neutral-buffered formalin, paraffin-embedded, routinely processed, andstained with hematoxylin and eosin. A board-certified pathologistevaluates the tissue sections for any evidence of tumor and/or teratomaformation which indicates successful induction of pluripotency.

Example 3 Karyotyping

iPSC clones generated in Example 1 are chosen for karyotyping. iPSCcells are plated in a 6-well plate and treated with 20 μl colcemid for 2hr in a 37° C. incubator to arrest the mitotic cells in metaphase.Colonies are lifted with ACCUTASE® (Stem Cell Technologies) andsubsequently centrifuged at 850 rpm for 3 min. The cell pellet issuspended in 0.067 M KCl hypotonic solution and incubated for 20 min atroom temperature. A 3:1 methanol:acetic acid fixative solution is addedto the hypotonic solution and incubated for 5 min at room temperature.This is followed by 3 rounds of treatment with a 3:1 methanol:aceticacid fixative solution, each round incubated for 1 hr at roomtemperature. Samples are dropped onto clean wet slides and aged in anoven at 90° F. for 1-2 hr. Afterwards, slides are immersed in trypsinfor 30-40 sec and rinsed in fetal bovine serum and saline. After anadditional rinse in saline, the slides are stained in 12.5% Giemsa inGurrs buffer for 2-3 min. Slides are rinsed in distilled water and airdried. Karotype analysis is then conducted on the slides.

Example 4 In Vitro Differentiation of Human iPSCs

iPSCs can be differentiated into any type of cell and protocols for suchdifferentiation are known to persons of ordinary skill in the art.

Embryoid bodies are generated from human iPSCs in suspension culture for6 days in medium with 15% KSR and then grown in adherent culture ongelatin-coated dishes with cytokine cocktails (e.g., 100 ng/ml SCF, 100ng.ml Flt3L, 50 ng/ml TPO, 100 ng/ml G-CSF, 20 ng/ml IGF-2, and 100ng/ml VEGF) to induce lymphoid lineage cells and cardiomyocytes.

For differentiation to dopaminergic neurons, iPSCs are cocultured withstromal cells in medium containing 10% KSR, 1×10⁻⁴ M non-essential aminoacids, and 2-mercaptoethanol for 16 days.

For induction of endoderm cells and pancreatic cells, iPSCs are culturedon feeder cells with 100 ng/ml activin A in medium supplemented with 2%FBS for 4 days and followed by an additional 8 days culture in mediumsupplemented with N2 and B-27 media supplements (both from ThermoFisherScientific), non-essential amino acids, β-mercaptoethanol, bovine serumalbumin, and L-glutamine.

Example 5 Assembly of a Library of iPSC Cell Lines

Cells from twenty cord blood units, each of which has a distincthaplotype that is homozygous at all of HLA-A, -B, and -DRB1 (see Table11) are treated to induce pluripotency as described above. The cells arepropagated to generate a seed stock for each. The seed stock cells arealiquoted and stored frozen in liquid nitrogen (LN₂). An aliquot fromthe seed stock is propagated to generate a working stock which isaliquoted and stored frozen in LN₂. An aliquot from the working stock ispropagated to generate library stock which is aliquoted and storedfrozen in LN₂.

Seed stock is used both to generate new working stock when existingworking stock becomes depleted and to replenish seed stock. Workingstock is used to replenish library stock when it becomes depleted.Quality control assays are conducted on samples from each stock as it isgenerated to confirm haplotype and insure the lack of contamination withother cell lines, or other organisms including for example, bacteria,yeast, and mold.

TABLE 11 Haplotypes of 20 Cord Blood Units from which an iPSC LineLibrary is Generated HLA LOCI A B DRB1 01:01 08:01 03:01 26:01 38:0104:02 03:01 07:02 15:01 29:02 44:03 07:01 02:01 44:02 04:01 02:01 35:0104:07 01:01 57:01 07:01 02:06 35:12 08:02 02:01 07:02 15:01 33:03 44:0307:01 24:02 52:01 15:02 01:01 35:02 11:04 30:01 13:02 07:01 33:03 58:0103:01 02:01 39:01 04:07 11:02 52:01 15:02 02:01 08:01 03:01 33:03 53:0108:04 26:01 38:01 13:02 01:01 57:01 13:05

More specifically, quality control assays are used to address five basicissues:

-   -   Identity, which must be repeatedly documented, and can be        assessed using methods such as STR, SNP and even genomic        sequencing.    -   Genomic Stability, which can be assessed by microarray methods        of karyotyping and CNV detection, nanostring technology, and        genomic sequencing.    -   Pluripotency, which can be assessed by marker expression,        embryoid body analysis and teratoma formation by        immunocytochemistry.    -   Persistence of expressionq of reprogramming factors, which can        be assessed by high sensitivity PCR assays.    -   Absence of contamination, which can be assessed using standard        microbiological assays for bacterial, fungal and mycoplasma in        culture.

Example 6 Demographic Comparison of the 20 Haplotypes

Using antigen-level resolution data for HLA-A and -B and allele-levelresolution data for DRB1 from the NMDP haplotype frequency and rankingwithin each of the four demographic groupings is compared. The librarycontains 9 of the 20 most common haplotypes for the Caucasian (CAU)population, 7 of the 20 most common haplotypes for the Hispanic (HIS)population, 6 of the of the 20 most common haplotypes for theAfrican-American (AFA) population, and 5 of the of the 20 most commonhaplotypes for the Asian & Pacific Islander (API) population. Thus, thelibrary has iPSC cell lines compatible with a meaningful portion of allfour of the defined demographic subpopulations.

TABLE 12 Haplotypes of 20 Cell Line Library for General Use for the USPopulation Showing Frequency and Ranking According to the NMDP Data SetHaplotype Frequency Ranking A B DRB1 CAU HIS AFA API CAU HIS AFA API 1 80301 0.062183 0.016801 0.011868 0.002979 1 2 2 36 1 35 1104 0.0026300.002399 0.000173 0.000640 40 55 1206 298 1 57 0701 0.011211 0.0042790.002373 0.010689 7 20 30 8 1 57 1305 0.001511 0.000066 0.0000440.000008 84 2087 3314 5227 2 7 1501 0.019904 0.005933 0.003798 0.0012394 11 12 141 2 8 0301 0.008890 0.003346 0.001748 0.000983 9 34 63 182 235 0407 0.000170 0.006589 0.000240 0.000189 862 10 882 945 2 35 08020.000152 0.015645 0.000181 0.000673 952 3 1161 278 2 39 0407 0.0000520.007890 0.000123 0.000110 2066 6 1615 1410 2 44 0401 0.020677 0.0036940.005146 0.000546 3 28 8 358 3 7 1501 0.030198 0.011575 0.0063850.002162 2 4 5 66 11 52 1502 0.001591 0.001966 0.000160 0.002523 77 771295 48 24 52 1502 0.000287 0.000297 0.000085 0.012177 577 609 2129 4 2638 0402 0.005399 0.001863 0.000221 0.000107 17 84 966 1432 29 44 07010.015507 0.018354 0.004269 0.000682 5 1 11 272 30 13 0701 0.0064850.003881 0.001190 0.011410 14 25 112 6 33 44 0701 0.000142 0.0008700.000473 0.016077 1023 190 440 2 33 53 0804 0.000022 0.000592 0.0072390.000021 3378 292 3 3527 33 58 0301 0.000236 0.000174 0.000443 0.019323668 1003 471 1

Based on the frequencies provided in Table 13 (and the figures woulddiffer if for example, allele-level resolution data were used for HAL-Aand/or HLA-B) 18.7%, 10.6%, 4.6%, and 8.2% of the CAU, HIS, AFA, and APIpopulations, respectively, have at least one of the 20 haplotypes. Butas discussed above, prospective recipients do not necessarily have tohave one of the 20 haplotypes. Rather their tissue type only needcontain an HLA-type or allele at each locus that matches the content ofthe haplotype. For example, the HLA-A1, B8, DRB1*0301 haplotype will bea match to persons whose tissue type contains any of the followinghaplotypes pairs:

1) HLA-A1, B8, DRB1*0301 plus anything;

2) HLA-A1, B8, DRB1*x plus HLA-Ax, Bx, DRB1*0301; and

3) HLA-A1, Bx, DRB1*0301 plus HLA-Ax, B8, DRB1*x,

4) HLA-A1, Bx, DRB1*x plus HLA-Ax, B8, DRB1*0301;

where x indicates any other the HLA-type or allele at that position. Thehaplotype frequencies given in Table 12 correspond to 1); 2) through 4)can further raise the likelihood of a match. However, due to thenon-random association of alleles the effect may be minimal. Table 12reports the frequencies of the haplotypes in 2) to 4) generated usingthe Haplotype Frequency Search function at the Allele Frequency NetDatabase.

TABLE 13 Frequencies of HLA-A1, B8, DRB1*03:01 related haplotypesHaplotype CAU HIS AFA API Pattern HLA-A HLA-B HLA-DRB1 (%) (%) (%) (%)(1) 01 08 03:01 6.2 1.7 1.2 0.3 (2) 01 08 x 1.4 0 0 0 x x 03:01 2.8 1.91.8 2.8 (3) 01 x 03:01 0 0 0 0 x 08 x 1.9 0.6 0.6 0.6 (4) 01 x x 11.01.3 0.4 2.4 x 08 03:01 1.9 0.6 0.3 0.6

From the data in Table 13 it can be seen that the pattern A*01, B*08,DRB1*x does not exist outside of the CAU population in any observedhaplotype except A*01, B*08, DRB1*03:01. Additionally the pattern A*01,B*x, DRB1*03:01 does not exist in any observed haplotype except A*01,B*08, DRB1*03:01 (starting from the A*01, B*08, DRB1*03:01 haplotype, itwould require two meiotic crossover events to generate this haplotypepattern). Thus haplotype pattern (2) contributes an additional frequencyof matching of 0.4% (multiply 1.4% by 2.8%) to the 6.2% frequency ofmatch from donor and prospective CAU recipient sharing the A*01, B*08,DRB1*03:01 haplotype. Haplotype pattern (3) is not expected to occur.Haplotype pattern (4) contributes an additional frequency of matching of0.2%, 0.01%, 0.001%, and 0.01% to the frequency of match from donor andprospective CAU, HIS, AFA, and API recipients sharing the A*01, B*08,DRB1*03:01 haplotype, respectively. Thus, the expected frequency ofmatching this haplotype is 6.8% for the CAU population but does notappreciably change for the other three populations. And in all cases thefrequency due to haplotype matching, rather than matching to the fulltissue type, provides a reasonable estimate of the likelihood ofmatching. This also illustrates that the primary gain in matchinglikelihood comes from avoiding the need to match two antigens at eachlocus. This pattern is a consequence of the substantial linkagedisequilibrium within the MHC. That tissue type matching will primarilyarise from a shared haplotype demonstrates the efficiency of thehomozygous donor approach.

TABLE 14 Frequencies of HLA-A2, B7, DRB1*15:01 related haplotypesHaplotype CAU HIS AFA API Pattern HLA-A HLA-B HLA-DRB1 (%) (%) (%) (%)(1) 02 07 15:01 2.0 0.6 0.4 0.1 (2) 02 07 x 0.8 0.1 0.2 0.1 x x 15:017.1 2.6 1.1 4.7 (3) 02 x 15:01 1.1 0.1 0.1 1.5 x 07 x 7.3 2.1 2.9 1.9(4) 02 x x 17.1 15.7 7.5 11.1 x 07 15:01 4.7 2.3 1.3 0.2

A similar analysis of the contribution to the likelihood of a match fromcomplementary, non-matching haplotypes, as done for A*01, B*08,DRB1*03:01, was also done for A*02, B*07, DRB1*15:01 (Table 14). As A2and B7 are the most common types for the HLA-A and -B loci in the CAUpopulation it appeared possible that they would be represented on a morediverse set of haplotypes. Also, as the CAU population is the leastgenetically diverse of the four subpopulations, the frequency ofindividual haplotypes will be on average higher and the contribution ofcomplementary, non-matching haplotypes to the overall likelihood of amatch will be generally greater. From the data in Table 13 it isimmediately apparent that there are observed haplotypes matching each ofthe haplotype patterns for all for subpopulations. The additionalmatching frequency for the tissue types with the haplotype pattern A*02,B*07, DRB1*x plus A*x, B*x, DRB1*15:01 was 0.06%, 0.003%, 0.002%, and0.005% for matching with prospective CAU, HIS, AFA, and API recipients,respectively. The additional matching frequency for the tissue typeswith the haplotype pattern A*02, B*x, DRB1*15:01 plus A*x, B*07, DRB1*xwas 0.08%, 0.002%, 0.003%, and 0.03% for matching with prospective CAU,HIS, AFA, and API recipients, respectively. The additional matchingfrequency for the tissue types with the haplotype pattern A*02, B*x,DRB1*x plus A*x, B*07, DRB1*15:01 was 0.8%, 0.4%, 0.1%, and 0.02% formatching with prospective CAU, HIS, AFA, and API recipients,respectively. Thus the expected frequency of having a tissue typematching this haplotype is 2.9% for the CAU population versus 2.0% forhaving the same haplotype. The same comparisons for HIS, AFA, and APIare 1.0 versus 0.6, 0.5 versus 0.4, and 0.16 versus 0.1, respectively.Thus even under favorable circumstances the additional matchingfrequency contributed by complementary, non-matching haplotypes is onlya fraction of that due to having the matching haplotype.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

What is claimed is:
 1. A library of HLA homozygous induced pluripotentstem cell (iPS) lines, the library comprising: at least 20 iPS celllines homozygous for alleles at HLA-A, -B, and -DR loci, each cell lineexpressing a different homozygous haplotype; wherein each of the iPScell lines has been reprogrammed into pluripotentiality by means ofnon-endogenous transcription factors.
 2. The library of claim 1, whereinsaid 20 iPS cell lines are homozygous additionally for alleles at one ofmore loci selected from HLA-C, -DQ, and DP,
 3. (canceled)
 4. The libraryof claim 1 wherein at least 15 of the 20 iPS cell lines carries ahaplotype comprising at least two alleles for a type selected from A1,A2, A3, A24, A29, A33, B7, B8, B35, B39, B44, B48, B52, B60, DR2, DR3,DR4, DR7, DR8, DR9, DR11, DR12, DR13, and DR15.
 5. (canceled)
 6. Thelibrary of claim 1, wherein each haplotype is compatible with a tissuetype of a fraction of the individuals in a defined population and thesum of fractions for all of the haplotypes in the library is at least 5,10, 15, 25, 30, 35, 40, 45, 50, 55, or 60 percent of the definedpopulation.
 7. The library of claim 6, wherein the library comprises allhaplotypes occurring at a frequency of >2%, >3% or >4% in the definedpopulation.
 8. The library of claim 6 wherein the library comprises≥50%, ≥60%, ≥70%, ≥30%, ≥90%, or ≥95% of all haplotypes occurring at afrequencyof >1%, >0.9%, >0.8%, >0.7%, >0.6%, >0.5%, >0.4%, >0.3%, >0.2%, >0.1%, >0.09%, >0.08, >0.07%, >0.06%or >0.05% in the defined population.
 9. The library of claim 6 whereinsaid defined population is defined by geographic origin, geographiclocation, ethnic background, or a combination thereof.
 10. The libraryof claim 9, wherein the defined geographical location is a state, acountry, a continent, or a region thereof.
 11. The library of claim 9,wherein the defined geographical location is the US or Japan. 12.(canceled)
 13. The library of claim 1, wherein the iPS cell lines arederived from cord blood. 14-15. (canceled)
 16. The library of claim 1wherein the at least 10 of the iPS cell lines have a haplotype selectedfrom the list of HLA-A* HLA-B* HLA-DRB1* 01:01 08:01 03:01 03:01 07:0215:01 02:01 44:02 04:01 02:01 07:02 15:01 29:02 44:03 07:01 02:01 15:0104:01 01:01 57:01 07:01 03:01 35:01 01:01 02:01 40:01 13:02 30:01 13:0207:01 02:01 08:01 03:01 02:01 57:01 07:01 24:02 07:02 15:01 11:01 35:0101:01 33:01 14:02 01:02 23:01 44:03 07:01 01:01 07:02 15:01 02:01 15:0113:01 02:01 13:02 07:01 31:01 40:01 04:04 25:01 18:01 15:01 02:01 44:0307:01 02:01 44:02 13:01 02:01 44:02 01:01 01:01 08:01 15:01 03:01 07:0201:01 02:01 44:02 15:01 02:01 51:01 11:01 26:01 38:01 04:02


17. The library of claim 1 wherein homozygous haplotypes expressed bythe at least 20 iPSC lines comprise A*01:01 B*08:01 DRB1*03:01 A*01:01B*35:02 DRB1*11:04 A*01:01 B*57:01 DRB1*07:01 A*01:01 B*57:01 DRB1*13:05A*02:01 B*07:02 DRB1*15:01 A*02:01 B*08:01 DRB1*03:01 A*02:01 B*35:01DRB1*04:07 A*02:06 B*35:12 DRB1*08:02 A*02:01 B*39:01 DRB1*04:07 A*02:01B*44:02 DRB1*04:01 A*03:01 B*07:02 DRB1*15:01 A*11:02 B*52:01 DRB1*15:02A*24:02 B*52:01 DRB1*15:02 A*26:01 B*38:01 DRB1*04:02 A*26:01 B*38:01DRB1*13:02 A*29:02 B*44:03 DRB1*07:01 A*30:01 B*13:02 DRB1*07:01 A*33:03B*044:03 DRB1*07:01 A*33:03 B*53:01 DRB1*08:04 A*33:03 B*58:01DRB1*03:01.


18. A method of producing a library of HLA homozygous iPSC comprising:screening cord blood units donated to a cord blood repository for: a)genetic, cytogenetic and other genomic and chromosomal abnormalities b)history and markers of exposure to infectious diseases c) homozygosityfor all of HLA-A, -B, -C, and -Dr loci; isolating white blood cells fromthe cord blood units negative for genetic defects, exposure toinfectious diseases, and transposons, and homozygous for HLA-A, -B, -C,and -Dr, causing the white blood cells to express Oct-4, Sox-2, Klf-4,and c-Myc to form iPS cells; culturing and cloning the iPSC to produce apopulation of cloned iPS cells homozygous for their HLA-A, -B, -C, and-DR haplotypes; characterizing the genotype of the iPSC by typing of HLAmarkers beyond the A,B,C and DR loci to include additional class I, IIand III genes; screening the cells from iPSC clones for the presence ofchromosomal aberrations and evidences of genetic instability andcryoprotecting and storing the cloned population of iPSC.
 19. The methodof claim 18, wherein the causing step includes using vectors capable ofinducing the target white blood cells to express the exogenoustranscription factors Oct-4, Sox-2, Klf-4, and c-Myc.
 20. (canceled) 21.The method of claim 18, wherein method further comprises isolatinghematopoietic CD34+ cells from the cord blood prior to the causing step.22-23. (canceled)
 24. A method for providing differentiated cellsderived from the iPSC library of claim 1 to a subject in need thereofcomprising: determining the HLA haplotype for HLA-A, -B, -C and -DR ofthe subject; selecting an iPSC line from the library of claim 1 whichcontains a match at all of HLA-A, -B, -C, and -DR loci with thesubject's HLA phenotype for those loci; differentiating the iPSC into acell type needed by the subject; and providing the differentiated cellsto the subject.
 25. The method of claim 24, wherein the step ofproviding the differentiated cells to the subject comprises providingthe differentiated cells to the subject's medical provider.
 26. Themethod of claim 24 further comprising expanding and fullydifferentiating the iPS cells into a differentiated cell type (e.g.,neural cells, myocardial muscle cells, insulin-producing cells, etc.)and ensuring the disappearance of the pluripotent cells and inducingvectors, as well as the genetic stability of the differentiated cells.27. The library of claim 1 wherein the at least 20 iPS cell lines arehomozygous for alleles at MICA and MICB loci, wherein the haplotypedifferences are at an HLA-A, -B, or -DR locus.
 28. The library of claim1 wherein the at least 20 iPS cell lines are homozygous for alleles atHLA-F and -DPB3 loci wherein the haplotype differences are at an HLA-A,-B, or -DR locus.